Natural regulatory T cells (nTreg) differentiate to acquire FoxpS expression and suppressive function in the thymus. These cells have been thought of as a highly stable lineage, but recent work suggests that they may lose Foxp3 expression and acquire various effector functions, particularly under conditions of lymphopenia and/or inflammation. It will be important to understand the conditions under which this loss may occur, if the expansion and transfer of nTreg, or in vivo augmentation of nTreg activity, will ever be used as immune therapies. We have made the surprising observation that nTreg transferred to Rag-/- mice require exogenous IL-10 from CD11b+ intestinal myeloid cells in order to prevent colitis. The lapsed nTreg in IL-10 deficient recipients acquire a distinct THI-like cytokine secretion pattern, characterized by the secretion of IFNy, but not TNF or IL-17. The experiments in this application therefore will use genetic technologies, cellular immunology methods and in vivo disease models to better understand the molecular changes and cellular interactions that underlie the striking in vivo changes in nTreg that we have observed. We will determine if the loss of nTreg function is reversible (Aim 1), and in Aim 2 we will fully characterize the function of the lapsed cells, and will determine if nTreg acting in other sites also require IL-10 from cells other than lymphocytes. Additionally, in this Aim we will examine the status ofthe Foxp3 locus in lapsed Treg, and the overall pattern of epigenetic modifications in their chromatin. We will determine in Aim 3 if induced Treg (ITreg) display a similar requirement for paracrine IL-10, and if the use of more effective induction methods, such as retinoic acid or alterations induced by viral infection, can fortify iTreg so they can function without exogenous IL-10. In aim 4, we will compare CD11b+ intestinal myeloid celis from different parts of the mucosal immune system for their ability to stimulate the generation of ITreg and the stabilization of nTreg. In summary, the proposed experiments build on our novel preliminary findings in order to achieve a deep understanding of the pathway leading to the dangerous loss of suppressive function in nTreg, and the means by which this can be reversed or prevented.